2-Heterosubstituted 3-aryl-4H-1-benzopyran-4-ones as novel therapeutics in breast cancer

ABSTRACT

The present invention provides 2-heterosubstituted 3-aryl-4H-benzopyran-4-one compounds for the treatment of cancers, namely breast cancer. This invention further provides a method of synthesis of 2-(alkylthio)isoflavones that can be carried out at ambient conditions. This invention further provides a method of synthesis of the 2-heterosubstituted 3-aryl-4H-benzopyran-4-one from a 2-(alkylthio)isoflavone. The invention further provides methods of using the 2-heterosubstituted 3-aryl-4H-benzopyran-4-one compounds for the treatment of breast cancer in mammals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/400,742, filed Aug. 2, 2002, the entirety of which isincorporated herein by reference.

STATEMENT ON FEDERALLY FUNDED RESEARCH

This work was supported, at least in part, by grants DMAD 17-00-1-0388and DMAD 17-99-1-9342 from the USAMRMC Breast Cancer Program. Thegovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to compounds, particularly2-heterosubstituted 3-aryl-4H-benzopyran-4-ones, for treating orpreventing cancer in a mammalian subject and methods of making and usingsuch 2-heterosubstituted 3-aryl-4H-benzopyran-4-ones.

BACKGROUND OF THE INVENTION

Breast cancer kills thousands of women annually. While surgicalintervention has saved the lives of many women, radical and partialmastectomies often prove physically and emotionally debilitating.Moreover, patients who have undergone surgery and subsequentchemotherapy often experience recurrence.

The 4H-1-benzopyran-4-one ring system is widely found in a number ofnatural products such as flavonoids. These natural products havedemonstrated numerous biological activities such as antiviral,anti-inflammatory, antiallergic, antimutagenic and anticarcinogenicactivities.¹ Genistein, an isoflavonoid, for example, has been known toact as an antagonist on the estrogen receptor suppressing thedevelopment of hormone-dependent breast cancer.

This fact has led us to investigate novel 4H-1-benzopyran-4-onederivatives as new therapeutic agents for hormone-dependent breastcancer.² As part of this effort, we have been interested in3-aryl-2-alkylthio-4H-1-benzopyran-4-ones, or 2-(alkylthio)isoflavones,as potential drug candidates in this area. However, only a few methodshave been reported for the synthesis of2-alkylthio-4H-1-benzopyran-4-ones.³ Furthermore, these methods sufferseveral disadvantages such as low yields, multiple steps, and harshconditions. Recently, a high yielding one-pot synthesis of2-methylthio-4H-1-benzopyranones has been reported using KHMDS as abase.⁴ However, this method also requires skillful handling of the base,low temperature, and anhydrous reaction conditions. A convenient methodof synthesis of 2-alkylthio-4H-1-benzopyran-4-ones or2-(alkylthio)isoflavones would provide a convenient starting point forthe development of new therapeutics for the treatment of breast cancer.

Accordingly, it is desirable to have new compounds and methods for thetreatment of hormone dependent breast cancer, as well as other cancers.It is also desirable to have new, efficient, methods for the synthesesof these new compounds. It is particularly desirable that these newsynthetic methods have few steps, improved yields, and mild conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a phase transfer catalyst procedure for the synthesis ofO-alkyl-S-methyl dithiocarbonates and a cyclization of theO-alkyl-S-methyl dithiocarbonates to generate the2-(alkylthio)isoflavones in a single step.

FIG. 2 shows the synthetic scheme for preparation of deoxybenzoins byFriedel-Crafts acylation of resorcinol with corresponding arylaceticacids followed by Mitsunobu reaction for the selective methylation of4-hydroxyl group.

FIG. 3 shows the production of a 2-(methylthio)isoflavone from thedeoxybenzoin.

FIG. 4 shows the production of five additional 2-(methylthio)isoflavonefrom three deoxybenzoins.

FIG. 5 shows dose response studies in MCF-7 cells for compounds 234,220, 244, and 230.

FIG. 6 shows dose responsive studies in MCF-7 cells for compounds 258,YWK-II-14, and 4-OH Tamoxifen.

FIG. 7 shows screening data for the isoflavone library (Example 3) inMCF-7 cells in the presence of Estradiol.

FIG. 8 shows screening data for the isoflavone library (Example 3) inMDA-MB-231 (ER−) cells.

FIG. 9 shows the synthetic scheme for the combinatorial librarycontaining compounds 308a–f, 309a–f, 310a–f, and 311a–f.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds for treating orpreventing cancer, particularly breast cancer in a subject. Thecompounds are new 2-heterosubstituted 3-aryl-4H-1-benzopyran-4-ones andpharmaceutical compositions that contain one of more of the2-heterosubstituted 3-aryl-4H-1-benzopyran-4-ones of the presentinvention and derivatives thereof. The 2-heterosubstituted3-aryl-4H-1-benzopyran-4-ones have structure A:

wherein

-   X is selected from the group consisting of O, N, S, SO, and SO₂;-   R₁ and R₂ can be the same or different and are selected from the    group consisting of H, OH, OCH₃, OCH₂CH₃, OCH₂C₆H₅, NH₂, NHCH₃,    N(CH₃)₂, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, C(CH₃)₃, NO₂, F, Cl, Br,    CF₃, SH, SCH₃, SCH₂CH₃, OCOCH3, OCOC(CH₃)₃, OCOCH₂COOH, and CN;-   R₃ is selected from the group consisting of H, OH, OCH₃, OCH₂CH₃,    NH₂, NHCH₃, N(CH₃)₂, NO₂, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, C(CH₃)₃,    F, Cl, Br, CF₃, SH, SCH₃, SCH₂CH₃, CN,

An especially preferred subclass of compounds are those of structure Awherein X is selected from S and O; R₁ is selected from OH and OC₆H₅; R₂is selected from OH and OCH₃; and R₃ is selected from OH and2-(1-piperidinyl)ethoxy.

In another aspect, the present invention provides a one-pot method ofmaking 2-(alkylthio)isoflavones. The method of synthesizing the2-(alkylthio)isoflavones comprises selecting a deoxybenzoin compound,preparing a mixture of the deoxybenzoin compound, carbon disulfide,alkyl halide, and tetrabutylammonium hydrogensulfate. While the mixtureis stirred, aqueous sodium hydroxide is added to the mixture. Theresulting mixture is allowed to stir until the reaction is complete,from about 3 to about 7 hours, at room temperature. Optionally, thedeoxybenzoin starting compound can be prepared by a Freidel-Craftsacylation of resorcinal with arylacetic acids followed by Mitsunobureaction. The resulting 2-(alkylthio)isoflavone can be filtered andpurified, if desired.

In another aspect of the present invention, the 2-(alkylthio)isoflavonecan then be used to prepare a 2-heterosubstituted3-aryl-4H-benzopyran-4-one. The method comprises (a) selecting a2-(alkylthio)isoflavone; (b) optionally protecting reactive groups onthe 2-(alkylthio)isoflavone; (c) oxidizing the alkylthio group to analkylsulfonyl group; and (d) substituting the alkylsulfonyl group with aheteroalkyl or heteroaryl group to form a 2-heterosubstituted3-aryl-4H-benzopyran-4-one. Preferably the oxidation step is carried outwith mCPBA in a polar, aprotic solvent, such as CH₂Cl₂. The methodfurther comprises the addition of drug-like substituents, such as1-piperidineylethoxy groups, on the 2-heteroaryl3-aryl-4H-benzopyran-4-ones. The method further comprises the step ofdeprotecting the 2-heteroaryl 3-aryl-4H-benzopyran-4-one compound. Themethod optionally further comprises a purification step.

In still another aspect of the present invention, a method of using thecompounds of formula A in the treatment of breast cancer, particularly,but not limited to hormone-dependent breast cancer. The method comprisesadministering a therapeutically effective amount of a2-heterosubstituted 3-aryl-4H-1-benzopyran-4-one compound of structure Ato a subject in need of treatment. The 2-heterosubstituted3-aryl-4H-1-benzopyran-4-one compound can be administered in accordancewith conventional methods, and in doses similar to drugs currentlyavailable for the treatment of breast cancer. The 2-heterosubstituted3-aryl-4H-1-benzopyran-4-one compounds of present invention may also beused as part of a combination therapy. The invention further provides amethod for the prevention of breast cancer in subjects who aresusceptible to developing breast cancer, comprising administering atherapeutically effective amount of a compound of structure A.

DETAILED DESCRIPTION OF THE INVENTION

The benzopyranone ring system is widely distributed in a number ofnatural products, including flavonoids. Compounds in this class haveshown a range of important biological and medicinal activities. We haveinvestigated a series of 2-alkyl substituted benzopyranone analogs toidentify potential drug candidates for hormone-dependent breast cancer.In the course of this study, we have also prepared heteroalkyl andheteroaryl benzopyranone analogs.

Our synthetic strategy for the construction of the2-alkylthiobenzopyranon-4-ones involves the simultaneous intramolecularcyclization of alpha-oxoketene dithioacetals derived from deoxybenzoinsas a key step under the TBS deprotonation condition. The alkylthio ethergroup at the 2 position can also serve as a good leaving group tointroduce other alkylated heteroatoms such as alkyl or aryl amines. Thissynthetic strategy is especially useful for solid-phase combinatorialsynthesis of medicinal agents for molecular targets in hormone-dependentbreast cancer.

The present invention provides 2-heterosubstituted3-aryl-4H-benzopyran-4-one compounds useful for the prevention andtreatment of cancers, particularly but not limited to breast cancers, aswell as methods of using these compounds to treat breast cancers insubjects in need of such treatment. The method involves treating thesubject in need of such treatment with a therapeutically effectiveamount of a 2-heterosubstituted 3-aryl-4H-benzopyran-4-one compound ofthe present invention or a derivative or pharmaceutically acceptablesalt thereof. The present invention also provides methods of making the2-heterosubstituted 3-aryl-4H-benzopyran-4-one compounds.

The 2-heterosubstituted 3-aryl-4H-benzopyran-4-one compounds and methodsof the present invention are useful for, but not limited to treating,inhibiting, or delaying the onset of cancers. The 2-heterosubstituted3-aryl-4H-benzopyran-4-one compounds of the present invention are alsouseful in the treatment of precancers. The compounds and methods areuseful for treating cancers including, but not limited to, breastcancer, leukemia, non-small cell lung cancer, colon cancer, CNS cancer,melanoma, ovarian cancer, renal cancer, prostate cancer, bladder cancer,and lymphoma. Furthermore, they are useful in the prevention of thesecancers in individuals with precancers, as well as individuals prone tothese disorders.

By “treating” is meant curing, ameliorating or tempering the severity ofthe cancer or the symptoms associated therewith. The terms “treating,”“treatment,” and “therapy” as used herein refer to curative therapy,prophylactic therapy, and preventative therapy.

“Preventing” or “prevention” means preventing the occurrence of thecancer, or tempering the severity of the cancer if it is developssubsequent to the administration of the instant compositions. Thispreventing the onset of a clinically evident unwanted cell proliferationaltogether or preventing the onset of a preclinically evident stage ofunwanted rapid cell proliferation in individuals at risk. Also intendedto be encompassed by this definition is the prevention of metastatis ofmalignant cells or to arrest or reverse the progression of malignantcells. This includes prophylactic treatment of those at risk ofdeveloping precancers and cancers.

The terms “therapeutically effective” and “pharmacologically effective”are intended to qualify the amount of each agent which will achieve thegoal of improvement in disease severity and the frequency of incidenceover treatment of each agent by itself, while avoiding adverse sideeffects typically associated with alternative therapies.

The term “subject” for purposes of treatment includes any human oranimal subject having a neoplasia, such as cancer or precancer. Formethods of prevention the subject is any human or animal subject, andpreferably is a human subject who is at risk of developing a cancer. Thesubject may be at risk due to exposure to carcinogenic agents, beinggenetically predisposed to disorders characterized by unwanted, rapidcell proliferation, and so on. Besides being useful for human treatment,the compounds of the present invention are also useful for veterinarytreatment of mammals, including companion animals and farm animals, suchas, but not limited to dogs, cats, horses, cows, sheep, and pigs.Preferably, subject means a human.

The term “derivative” is intended to encompass compounds which arestructurally related to the present invention or which possess thesubstantially equivalent activity to the parent 2-heterosubstituted3-aryl-4H-benzopyran-4-one compounds, as measured by the derivative'sability to inhibit activity in an in vitro estrogen dependent cellproliferation assay using human breast cells (MCF-7). By way of example,such compounds may include, but are not limited to, prodrugs thereof.Such compounds can be formed in vivo, such as by metabolic mechanisms.

Where the term alkyl is used, either alone or with other terms, such ashaloalkyl or alkylaryl, it includes C₁ to C₁₀ linear or branched alkylradicals, examples include methyl, ethyl, propyl, isopropyl, butyl,tert-butyl, and so forth. The term “haloalkyl” includes C₁ to C₁₀ linearor branched alkyl radicals substituted with one or more halo radicals.Some examples of haloalkyl radicals include trifluoromethyl,1,2-dichloroethyl, 3-bromopropyl, and so forth. The term “halo” includesradicals selected from F, Cl, Br, and I.

The term aryl, used alone or in combination with other terms such asalkylaryl, haloaryl, or haloalkylaryl, includes such aromatic radicalsas phenyl, biphenyl, and benzyl, as well as fused aryl radicals such asnaphthyl, anthryl, phenanthrenyl, fluorenyl, and indenyl on so forth.The term “aryl” also encompasses “heteroaryls,” which are aryls thathave carbon and one or more heteroatoms, such as O, N, or S in thearomatic ring. Examples of heteroaryls include indolyl, pyrrolyl, and soon. “Alkylaryl” or “arylalkyl” refers to alkyl-substituted aryl groupssuch as butylphenyl, propylphenyl, ethylphenyl, methylphenyl,3,5-dimethylphenyl, tert-butylphenyl and so forth.

The agents of the present invention may be administered orally,intravenously, intranasally, rectally, or by any means which delivers aneffective amount of the active agent to the tissue or site to betreated. It will be appreciated that different dosages may be requiredfor treating different disorders. An effective amount of an agent isthat amount which causes a statistically significant decrease inneoplastic cell count, growth, or size. Neoplastic disorders responsiveto the agents of the present invention include, but are not limited to,breast cancer.

The dosage form and amount can be readily established by reference toknown treatment or prophylactic regiments. The amount of therapeuticallyactive compound that is administered and the dosage regimen for treatinga disease condition with the compounds and/or compositions of thisinvention depends on a variety of factors, including the age, weight,sex, and medical condition of the subject, the severity of the disease,the route and frequency of administration, the particular compoundemployed, the location of the unwanted proliferating cells, as well asthe pharmacokinetic properties of the individual treated, and thus mayvary widely. The dosage will generally be lower if the compounds areadministered locally rather than systemically, and for prevention ratherthan for treatment. Such treatments may be administered as often asnecessary and for the period of time judged necessary by the treatingphysician. One of skill in the art will appreciate that the dosageregime or therapeutically effective amount of the inhibitor to beadministrated may need to be optimized for each individual. Thepharmaceutical compositions may contain active ingredient in the rangeof about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mgand most preferably between about 1 and 200 mg. A daily dose of about0.01 to 100 mg/kg body weight, preferably between about 0.1 and about 50mg/kg body weight, may be appropriate. The daily dose can beadministered in one to four doses per day.

The active agents may be administered along with a pharmaceuticalcarrier and/or diluent. The agents of the present invention may also beadministered in combination with other agents, for example, inassociation with other chemotherapeutic or immunostimulating drugs ortherapeutic agents. Examples of pharmaceutical carriers or diluentsuseful in the present invention include any physiological bufferedmedium, i.e., about pH 7.0 to 7.4 comprising a suitable water solubleorganic carrier. Suitable water soluble organic carriers include, butare not limited to corn oil, dimethylsulfoxide, gelatin capsules, etc.

Also included in the family of 2-heterosubstituted3-aryl-4H-benzopyran-4-one compounds are the pharmaceutically acceptablesalts thereof. The phrase “pharmaceutically acceptable salts” connotessalts commonly used to form alkali metal salts and to form additionsalts of free acids or free bases. The nature of the salt is notcritical, provided that it is pharmaceutically acceptable.

Suitable pharmaceutically acceptable acid addition salts of2-heterosubstituted 3-aryl-4H-benzopyran-4-one compounds may be preparedfrom an inorganic acid or from an organic acid. Examples of suchinorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric,carbonic, sulfuric, and phosphoric acid. Appropriate organic acids maybe selected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic, and sulfonic classes of organic acids,examples of which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic,salicylic, p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic,methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, galactaric,and galacturonic acids.

Suitable pharmaceutically acceptable base addition salts of2-heterosubstituted 3-aryl-4H-benzopyran-4-one compounds includemetallic salts made from aluminum, calcium, lithium, magnesium,potassium, sodium, and zinc. Alternatively, organic salts made fromN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine may be usedform base addition salts of the 2-heterosubstituted3-aryl-4H-benzopyran-4-one compounds. All of these salts may be preparedby conventional means from the corresponding 2-heterosubstituted3-aryl-4H-benzopyran-4-one compounds by reacting, for example, theappropriate acid or base with the 2-heterosubstituted3-aryl-4H-benzopyran-4-one compound.

The phrase “adjunct therapy” (or “combination therapy”), in defining useof a compound of the present invention and one or more otherpharmaceutical agent, is intended to embrace administration of eachagent in a sequential manner in a regimen that will provide beneficialeffects of the drug combination, and is intended as well to embraceco-administration of these agents in a substantially simultaneousmanner, such as in a single formulation having a fixed ratio of theseactive agents, or in multiple, separate formulations for each agent.

There are large numbers of antineoplastic agents available in commercialuse, in clinical evaluation and in pre-clinical development, which couldbe selected for treatment of cancers or other neoplasias by combinationdrug chemotherapy. Such antineoplastic agents fall into several majorcategories, namely, antibiotic-type agents, alkylating agents,antimetabolite agents, hormonal agents, immunological agents,interferon-type agents and a category of miscellaneous agents.Alternatively, other anti-neoplastic agents, such as metallomatrixproteases inhibitors may be used. Suitable agents which may be used incombination therapy will be recognized by those of skill in the art.

For oral administration, the pharmaceutical composition may be in theform of, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a particular amount of the active ingredient. Examplesof such dosage units are capsules, tablets, powders, granules or asuspension, with conventional additives such as lactose, mannitol, cornstarch or potato starch; with binders such as crystalline cellulose,cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators such as corn starch, potato starch or sodiumcarboxymethyl-cellulose; and with lubricants such as talc or magnesiumstearate. The active ingredient may also be administered by injection asa composition wherein, for example, saline, dextrose or water may beused as a suitable carrier.

For intravenous, intramuscular, subcutaneous, or intraperitonealadministration, the compound may be combined with a sterile aqueoussolution which is preferably isotonic with the blood of the recipient.Such formulations may be prepared by dissolving solid active ingredientin water containing physiologically compatible substances such as sodiumchloride, glycine, and the like, and having a buffered pH compatiblewith physiological conditions to produce an aqueous solution, andrendering said solution sterile. The formulations may be present in unitor multi-dose containers such as sealed ampoules or vials.

Synthetic Methods Several years ago a phase transfer catalysis procedurewas reported for the synthesis of O-alkyl-S-methyl dithiocarbonates (3)in high yields (FIG. 1, Scheme 1).⁵ We investigated this procedure forthe synthesis of 2-(alkylthio)isoflavones (2) from deoxybenzoins. Thepurpose of our study was to determine whether the O-aryl-S-alkyldithiocarbonates 3′ would undergo further cyclization reaction in thisreaction condition, thereby directly generating the desired2-(alkylthio)isoflavones 2 in a single step (FIG. 1, Scheme 2).

EXAMPLE 1 General Experimental Procedure for Preparing2-(alkylthio)isoflavones

To a stirred mixture of a deoxybenzoin (1 mmol), carbon disulfide (0.6mL, 10 mmol), alkyl halide (2.2 mmol), and tetrabutylammoniumhydrogensulfate (34 mg, 0.1 mmol) in THF (3 mL) and water (1 mL) wasslowly added aqueous NaOH solution (1.2 mL, 12 mmol, 10 M) at roomtemperature. A slight exothermic reaction and a color change of themixture were observed. The resulting mixture was vigorously stirred atroom temperature for several hours, and the product was extracted withethyl acetate (2×10 mL). The separated organics were washed with water(10 mL) and then with brine (10 mL), dried over MgSO₄, and filtered. Thefiltrate was concentrated under reduced pressure, and the residue waspurified by silica gel column chromatography (eluting with EtOAc/hexaneor MeOH/CHCl₃) to give the product as a white solid. All the productswere recrystallized from EtOAc/hexane.⁹

EXAMPLE 2 Preparation of the Deoxybenzoins

Deoxybenzoins 6 were prepared by Friedel-Crafts acylation of resorcinolwith corresponding arylacetic acids followed by Mitsunobu reaction forthe selective methylation of 4-hydroxyl group (FIG. 2, Scheme 3).⁶

When the deoxybenzoin 6a, carbon disulfide, and methyl iodide in aTHF⁷-water two-phase system were treated with aqueous NaOH solution atroom temperature in the presence of 10 mole % of tetrabutylammoniumhydrogensulfate (n-Bu₄N.HSO₄), the desired 2-(methylthio)isoflavone 2awas obtained in 87% yield (FIG. 3, Scheme 4). The reaction apparentlyoccurred in a stepwise manner as monitored by TLC. Interestingly, thespectral data of the intermediate indicated that the reaction proceedednot via the O-alkyl-S-methyl dithiocarbonate but via α-oxoketenedithioacetal (7).⁸ This could be explained by the intramolecularhydrogen bond of the starting deoxybenzoin, which might facilitate theenolate formation. In addition, the formation of α-oxoketenedithioacetal intermediate (FIG. 3, step A, Scheme 4) is very fast; allthe starting material was converted into the intermediate as soon as theaqueous NaOH solution was added. However, when the reaction is done at0° C., intermediate 7 can be obtained within 5 min in a high yield(>90%). In contrast, the subsequent cyclization (FIG. 3, step B, Scheme4) is the rate-determining step, and thereby requires a longer reactiontime (3–4 h) to be completed presumably because the intramolecularhydrogen bond of intermediate 7 is still rigid. However, this reactioncan be completed within an hour when the reaction mixture is warmed at˜50° C.

Similar results were obtained when deoxybenzoin 6b and 6c were used asstarting materials (FIG. 4, Scheme 5). Therefore, this method proved tobe efficient to prepare the 2-(methylthio)isoflavones, which can be usedas useful intermediates to introduce various nucleophiles at the2-position. In addition, as shown in Scheme 5 (FIG. 4), this process wasalso efficient when different alkyl halides were used as an alkylatingagent, indicating diverse alkylthio groups can be directly introduced atthe 2-position using this process.

In summary, we have described a convenient phase transfer catalysisprocedure allowing the efficient conversion of deoxybenzoins into2-(alkylthio)isoflavones in a single step at the ambient reactioncondition. This method can be very useful to generate a number ofdrug-like compounds from easily available deoxybenzoins in a shortperiod of time.

EXAMPLE 3 Preparation of the 2-heterosubstituted3-aryl-4H-1-benzopyran-4-ones

The 2-heterosubstituted 3-aryl-4H-1-benzopyran-4-one compounds areformed from the 2-(alkylthio)isoflavones of Example 1 by the addition ofa side chain as the 2-position through oxidation then substitution ofthe methylthio group. Reflux of the 2-(alkylthio)isoflavone with mCPBAin CH₂Cl₂ for approximately one hour resulted in the oxidation of thestarting 2-(alkylthio)isoflavone compound. The substitution at the2-position was achieved by mixing the oxidized 2-(alkylthio)isoflavonewith p-sulfhydrylphenol, and sodium hydride in DMF for 15 minutes atroom temperature. Then, the 2-substituent of the resulting compound wasconverted to a 4-[2-(1-piperidinyl)ethoxy]thiophenoxy group by reactingwith of 2-piperidinylchloroethane and HCl in Cs₂CO₃, DMF for 8 hours atroom temperature to form the end product. Subsequent deprotection wascarried out with BBr₃, less than 2 eq. CH₂Cl₂ to yield hydroxyl groupsat the R₁ and R₂ positions. Alternatively, BF₃OEt₂ and Me₂S, both inexcess in CH₂Cl₂ was used to give hydroxyl groups at the R₁, R₂, and R₃positions.

EXAMPLE 4

Several members of the combinatorial library that was prepared inaccordance with the methods of this invention have been tested foreffects on proliferation in MCF-7 cells and MDA-MB-231 cells. Theresults are shown in Table 1.

TABLE 1

Compound X R₁ R₂ R₃ M.F. M.W. YWK-I-220 S OBn OMe OH C₂₉H₂₂O₅S 482.55YWK-I-244 S OH OH OH C₂₁H₁₄O₅S 378.40 YWK-I-230 S OBn OMe2-(1-piperidinyl)ethoxy C₃₆H₃₅NO₅S HCl 630.19 YWK-I-260 S OH OH2-(1-piperidinyl)ethoxy C₂₈H₂₇NO₅S 489.58 YWK-I-234 S OH OMe2-(1-piperidinyl)ethoxy C₂₉H₂₉NO₅S 503.61 YWK-I-246 O OBn OMe OHC₂₉H₂₂O₆ 466.48 YWK-I-268 O OH OH OH C₂₁H₁₄O₆ 362.33 YWK-I-258 O OBn OMe2-(1-piperidinyl)ethoxy C₃₆H₃₅NO₆HCl 614.13 YWK-I-262 O OH OH2-(1-piperidinyl)ethoxy C₂₈H₂₇NO₆ 473.52 YWK-II-14 O OH OMe2-(1-piperidinyl)ethoxy C₂₉H₂₉NO₆ 487.54

EXAMPLE 5

Dose response studies were performed with compounds from thecombinatorial library. Compounds 234, 220, 244, 230, 258, YWK-II-14 and4-OH-Tamoxifen were performed. The responses, plotted as Percent ofControl versus log [concentration/nM] is shown in FIGS. 5 and 6.

EXAMPLE 6

IC₅₀ ranges for several members of the combinatorial library weredetermined within 95% confidence intervals. The results are shown inTable 2, below.

TABLE 2 IC₅₀ ± ΔIC₅₀ IC₅₀ (μM) Compound # IC₅₀ Range (μM) (From Program)R² 174 — — — — 234 0.36–0.92 0.64 ± 0.28 0.58 .904 244  7.86–15.74 11.80± 3.94  11.12 .916 260 — — — — 262 — — — — 220 2.87–7.68 5.28 ± 2.404.70 .955 230 2.41–7.23 4.82 ± 2.41 4.17 .940 246 — — — — 258 2.30–5.884.09 ± 1.79 3.67 .900 268 — — — — YWK-II-14 6.31* 6.31* 6.31* .994 4-OHTamoxifen 3.61–4.93 4.27 ± 0.66 4.22 .993 —= Compounds that did notinhibit 50%, even at high concentrations; *= The value is an estimationdue to problems analyzing the data using GraphPad Prism

EXAMPLE 7

Screening assays were done with the compounds from the combinatoriallibrary in MC-7 (ER+) Cells in the presence of Estradiol. The results ofthe screening are shown graphically in FIG. 7.

EXAMPLE 8

Screening assays for cytotoxicity (2-day) and proliferation (5 day)against the MDA-MB-231 (ER−) cells were preformed. The results aredisplayed graphically in FIG. 8.

EXAMPLE 9

The following combinatorial compounds were all tested in cell with goodactivity.

TABLE 3 Compounds tested in Cell, all compounds were soluble in DMSO

Amount Com- Mol. (mg), 10 pound X R₁ R₂ R₃ Wt. μmol each YWK-I- S OH OHOH 378.40 3.78 244 YWK-I- S OH OH 2-1- 489.58 490 260(piperidinyl)ethoxy YWK-I- S OH OMe 2-1- 503.61 5.04 234(piperidinyl)ethoxy YWK-I- O OH OH OH 362.33 3.62 268 YWK-I- O OH OH2-1- 473.52 4.74 262 (piperidinyl)ethoxy YWK-I- O OH OMe 2-1- 487.544.88 272 (piperidinyl)ethoxy

EXAMPLE 10

The following combinatorial library (compounds 308a–f to 311a–f) wasprepared and tested.

TABLE 4

Compound X R₁ R₂ % Yield 308a S OMe OBn 96 308b O OMe OBn 93 308c S HOMe 90 308e S Me OMe 83 308f S OMe OMe 94

TABLE 5

Compound X R₁ R₂ % Yield 309a S OMe OBn 83 309b O OMe OBn 89 309c S HOMe 84 309d S Me OMe 73 309e S OMe OMe 78

TABLE 6

Compound X R₁ % Yield 310a S OMe 86 310b O OMe 87 310c S OH 93 310d O OH92 310e S H 85 310f S Me 81

TABLE 7

Compound X R₁ % Yield 311a S OMe 89 311b O OMe 88 311c S OH 83 311d O OH75 311e S H 78 311f S Me 73Thus, we designed and synthesized several isoflavones containing a basicside chain connected through either an oxygen or a sulfur linkage(synthetic scheme—FIG. 9). Several isoflavones in this series showedpotent activities in suppressing proliferation of human breast cancercell lines, among which compound 310a is the most potent (IC₅₀=0.058μM). Interestingly, however, none of these compounds showed significantbinding affinities for ERs, indicating their potent antiproliferativeactivities might be mediated by other mechanisms.

All documents referenced herein are incorporated by reference.

Although this invention has been described with respect to specificembodiments, the details of these embodiments are not to be construed aslimitations.

REFERENCES

-   1. (a) Cassady, J. M.; Zennie, T. M.; Chae, Y. H. Cancer Res. 1988,    48, 6257; (b) Collins, M. B.; Mclachlan, J. A.; Arnold, S. F.    Steroids 1997, 62, 365; (c) Martin, P. M.; Horowitz, K. B.;    Ryan, D. S. Endocrinology 1978, 103, 1860; (d) Vrijsen, R.;    Everaert, L.; Boeye, A. J. Gen. Virol. 1988, 69, 1749; (e)    Dickancaite, E.; Nemeikaite, A.; Kalvelyte, A. Biochem. Mol. Biol.    Int. 1998, 45, 923; (f) Shao, Z. M.; Wu, J.; Shen, Z. Z. Cell.    Biochem. 1998, 69, 44.-   2. (a) Bhat, A. S.; Whetstone, J. L.; Brueggemeier, R. W.    Tetrahedron Lett. 1999, 40, 2469; (b) Brueggemeier, R. W.;    Richards, J. A.; Joomprabutra, S.; Bhat, A. S.; Whetstone, J. L. J.    Steroid Biochem. Mol. Biol. 2001, 79, 75.-   3. (a) Eiden, F.; Schünemann, J. Arch. Pharm. (Weinheim) 1985, 318,    1096; (b) Bantick, J. R.; Suschitzky, J. L. J Heterocyclic Chem.    1981, 18, 679.-   4. Lee, G. H.; Pak, C. S. Synth. Commun. 1999, 29, 2539.-   5. Lee, A. W. M.; Chan, W. H.; Wong, H. C.; Wong, M. S. Synth.    Commun. 1989, 19, 547.-   6. (a) For the reaction conditions for the synthesis of compounds 5,    see: Wähälä, K.; Hase, T. A. J. Chem. Soc., Perkin Trans. 1 1991,    3005; (b) Compounds 6a and 6c are commercially available.; (c)    Physical and spectral data for compound 6b: mp 71–72° C.; IR (KBr)    1639, 1623, 1516, 1508, 1439, 1388, 1355, 1268, 1231, 1205, 1131    cm⁻¹; ¹H NMR (250 MHz, CDCl₃) δ 12.72 (s, 1H), 7.73 (d, J=8. 6 Hz,    1H), 7.14–7.24 (m, 4H), 6.43–6.40 (m, 2H), 4.15 (s, 2H), 3.81 (s,    3H), 2.31 (s, 3H); ¹³C NMR (62.9 MHz, CDCl₃) δ 202.63, 166.56,    166.28, 137.14, 132.47, 131.71, 129.87, 129.61, 113.60, 108.20,    101.43, 55.98, 44.89, 21.48; HRMS calcd for C₁₆H₁₆NaO₃ (M+Na)⁺    279.0997, found 279.0989.-   7. We have modified the original reaction condition by using THF as    an organic solvent instead of carbon disulfide.-   8. NMR data for compound 7: ¹H NMR (400 MHz, CDCl₃) δ 6 12.37 (s,    1H), 7.56 (d, J=8.6 Hz, 1H), 7.43–7.45 (m, 2H), 7.27–7.35 (m, 3H),    6.39–6.43 (m, 2H), 3.80 (s, 3H), 2.27 (s, 3H), 2.25 (s, 3H); ¹³C NMR    (100 MHz, CDCl₃) δ 199.21, 166.71, 166.46, 143.82, 137.06, 136.34,    134.11, 129.21, 128.96, 128.83, 113.88, 108.40, 101.43, 56.03,    18.05, 17.28.-   9. Physical and spectral data for compounds 2: (a) 2a: mp 140–142°    C.; IR (KBr) 1629, 1619, 1584, 1541, 1499, 1431, 1373, 1349, 1252,    1201 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.13 (d, J=8.9 Hz, 1H),    7.32–7.44 (m, 5H), 6.96 (dd, J=8.9, 2.4 Hz, 1H), 6.84 (d, J=2.3 Hz,    1H), 3.91 (s, 3H), 2.53 (s, 3H); ¹³C NMR (62.9 MHz, CDCl₃) δ 173.96,    164.41, 164.01, 158.52, 132.64, 131.05, 128.88, 128.68, 128.34,    122.29, 117.66, 114.62, 100.10, 56.30, 14.12; HRMS calcd for    C₁₇H₁₅O₃S (M+H)⁺ 299.0742, found 299.0735. (b) 2b: mp 188–189.5° C.;    IR (KBr) 1632, 1612, 1544, 1510, 1433, 1368, 1287, 1265 cm⁻¹; ¹H NMR    (400 MHz, CDCl₃) δ 8.14 (d, J=8.9 Hz, 1H), 7.20–7.26 (m, 4H), 6.95    (dd, J=8.9, 2.3 Hz, 1H), 6.83 (d, J=2.3 Hz, 1H), 3.90 (s, 3H), 2.53    (s, 3H), 2.37 (s, 3H); ¹³C NMR (62.9 MHz, CDCl₃) δ 174.08, 164.20,    163.96, 158.51, 138.50, 130.84, 129.68, 129.58, 128.36, 122.24,    117.66, 114.55, 100.08, 56.28, 21.85, 14.14; HRMS calcd for    C₁₈H₁₆NaO₃S (M+Na)⁺ 335.0718, found 335.0721. (c) 2c: mp 169–170°    C.; IR (KBr) 1616, 1540, 1437, 1374, 1348, 1252 cm⁻¹; ¹H NMR (400    MHz, CDCl₃) δ 8.12 (d, J=8.8 Hz, 1H), 7.24–7.27 (m, 2H), 6.93–6.96    (m, 3H), 6.83 (d, J=2.3 Hz, 1H), 3.90 (s, 3H), 3.82 (s, 3H), 2.52    (s, 3H); ¹³C NMR (62.9 MHz, CDCl₃) δ 174.15, 164.31, 163.95, 159.90,    158.50, 132.24, 128.32, 124.69, 121.82, 117.61, 114.57, 114.40,    100.07, 56.28, 55.65, 14.15; HRMS calcd for C₁₈H₁₆NaO₄S (M+Na)⁺    351.0667, found 351.0668. (d) 2d: mp 117–118° C.; IR (KBr) 1635,    1615, 1585, 1546, 1503, 1435, 1373, 1345, 1252, 1197 cm⁻¹; ¹H NMR    (400 MHz, CDCl₃) δ 8.13 (d, J=8.9 Hz, 1H), 7.30–7.44 (m, 5H), 6.96    (dd, J=8.9, 2.4 Hz, 1H), 6.81 (d, J=2.3 Hz, 1H), 5.83–5.94 (m, 1H),    5.27 (dd, J=16.9, 1.2 Hz, 1H), 5.14 (dd, J=10.1, 0.8 Hz, 1H), 3.91    (s, 3H), 3.70 (d, J=6.9 Hz, 2H); ¹³C NMR (62.9 MHz, CDCl₃) δ 174.21,    164.12, 163.40, 158.50, 133.20, 132.61, 131.08, 128.82, 128.68,    128.39, 123.35, 119.27, 117.69, 114.51, 100.13, 56.31, 34.54; HRMS    calcd for C₁₉H₁₆NaO₃S (M+Na)⁺ 347.0718, found 347.0705. (e) 2e: mp    153–154° C.; IR (KBr) 1636, 1617, 1586, 1546, 1502, 1438, 1373,    1341, 1252, 1205 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.12 (d, J=8.9 Hz,    1H), 7.22–7.41 (m, 10H), 6.95 (dd, J=8.9, 2.4 Hz, 1H), 6.81 (d,    J=2.3 Hz, 1H), 4.30 (s, 2H), 3.91 (s, 3H); ¹³C NMR (62.9 MHz, CDCl₃)    δ 174.22, 164.10, 163.54, 158.48, 136.51, 132.52, 131.04, 129.32,    129.17, 128.81, 128.66, 128.39, 128.17, 122.97, 117.70, 114.49,    100.19, 56.31, 36.17; HRMS calcd for C₂₃H₁₈NaO₃S (M+Na)⁺ 397.0874,    found 397.0856.

1. A compound of formula A:

wherein X is selected from the group consisting of O, N, S, SO, and SO₂;R₁ and R₂ can be the same or different and are selected from the groupconsisting of H, OH, OCH₃, OCH₂CH₃, OCH₂CH₅, NH₂, NHCH₃, N(CH₃)₂, CN,CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, C(CH₃)₃, NO₂, F, Cl, Br, CF₃, SH,SCH₃, SCH₂CH₃, OCOCH3, OCOC(CH₃)₃, and OCOCH₂COOH; R₃ is


2. A compound of formula A:

wherein X is selected from S, N, and O; R₁ and R₂ can be the same ordifferent and are selected from the group consisting of H, OH, OCH₃,OCH₂H₃, CCH₂C₆H₅, NH₂, NHCH₃, N(CH₃)₂, CN, CH₃, CH₂CH₃, CH₂CH₂CH₃,CH(CH₃)₂, C(CH₃)₃, NO₂, F, Cl, Br, CF₃, SH, SCH₃, SCH₂CH₃, OCOCH₃,OCOC(CH₃)₃, and OCOCH₂COOH; and R₃ is 2-(1-piperidinyl)ethoxy.
 3. Thecompound of claim 2, wherein X is S, R₁ is OH, R₂ is OCH₃, and R₃ is2-(1-piperidinyl)ethoxy.
 4. The compound of claim 2 wherein X isselected from S and O; R₁ is selected from OH, OCH₃, and OC₆H₅; R₂ isselected from H, OH, CH₃, and OCH₃; and R₃ is 2-(1-piperidinyl)ethoxy.5. The compound of claim 2, wherein X is S, R₁ is OC₆H₅, R₂ is OCH₃, andR₃ is 2-(1-piperidinyl)ethoxy.
 6. The compound of claim 2, wherein X isO, R₁ is OC₆H₅, R₂ is OCH₃, and R₃ is 2-(1-piperidinyl)ethoxy.
 7. Thecompound of claim 2, wherein X is O, R₁ is OH, R₂ is OCH₃, and R₃ is2-(1-piperidinyl)ethoxy.
 8. A one-pot method for preparing a2-(alkylthio)isoflavone comprising the steps of: a. providing a mixtureof a deoxybenzoin, carbon disulfide, alkyl halide, andtetrabutylammonium hydrogensulfate; b. adding aqueous sodium hydroxideto the mixture while stirring; c. reacting the mixture until the2-(alkylthio)isoflavone is formed.
 9. The method of claim 8 wherein themixture is allowed to stir for about 3 to about 7 hours after theaddition of the sodium hydroxide.
 10. The method of claim 8 furthercomprising the step of separating the 2-(alkylthio)isoflavone from thereaction mixture.
 11. The method of claim 10 further comprising the stepof purifying the 2-(alkylthio)isoflavone compound.
 12. A method ofprepararing a 2-heterosubstituted 3-aryl-4H-benzopyran-4-one compoundcomprising the steps of: a. selecting a 2-(alkylthio)isoflavone; b.optionally protecting potentially reactive groups on the2-(alkylthio)isoflavone; c. oxidizing the alkylthio group to aalkylsyfonyl group; and d. substituting the alkylsulfonyl group with aheteroalkyl or heteroaryl group to form the 2-heterosubstituted3-aryl-4H-benzopyran-4-one compound.
 13. The method of claim 12 whereinthe oxidation step is carried out using mCPBA in a polar aprotic solventunder reflux conditions.
 14. The method of claim 12 wherein the polaraprotic solvent is CH₂Cl₂.
 15. The method of claim 12 whereinalkylsulfonyl group is substituted with a thioaryl group.
 16. The methodof claim 15 further comprising the step of substituting the thioarylgroup with an ethylpiperidinyl group to form a4-[2-(1-piperidinyl)ethoxy]thiophenyl group at the 2-position of the2-heterosubstituted 3-aryl-4H-benzopyran-4-one compound.
 17. The methodof claim 16 further comprising the step of deprotecting the2-heterosubstituted 3-aryl-4H-benzopyran-4-one.
 18. The method of claim12 further comprising the step of deprotecting the 2-heterosubstituted3-aryl-4H-benzopyran-4-one.
 19. A method for treating, inhibiting, ordelaying the onset of a breast cancer in a subject in need of suchtreatment; the method comprising administering a therapeuticallyeffective amount of compound A:

wherein X is selected from the group consisting of O, N, S, SO, and SO₂;R₁ and R₂ can be the same or different and are selected from the groupconsisting of H, OH, OCH₃, OCH₂CH₃, OCH₂C₆H₅, NH₂, NHCH₃, N(CH₃)₂, CN,CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, C(CH₃)₃, NO₂, F, Cl, Br, CF₃, SH,SCH₃, SCH₂CH₃, OCOCH3, OCOC(CH₃)₃, and OCOCH₂COOH; and R₃ is2-(1-piperidinyl)ethoxy to the subject in need of such treatment. 20.The method of claim 19 wherein the cancer is hormone-dependent breastcancer.